Throughout this application various publications are referred to in parenthesis. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
The development of blood substitutes as in vivo oxygen-carriers has been one of the major aspects of modern transfusion medicine (1). The limitations of acellular hemoglobin (Hb) to be used as Hb based oxygen carriers, such as low plasma retention time, and the high oxygen affinity (relative to that of erythrocytes) were addressed in the design of the first generation of blood substitute products. The preclinical and clinical investigations of the first generation products have identified the vasoconstrictive (hyper-tension) activity of acellular Hb (inducing a ‘pressor’ effect) as the major toxicity of the first generation products (2–8). Accordingly, intense efforts have been directed over the years to overcome this limitation and/or the toxicities of acellular Hb by chemical modification of hemoglobin or by a combination of site directed mutagenesis and chemical modification (9–18).
The intrinsic high affinity of Hb for nitric oxide (NO) (a vasodilator) has been advanced as the molecular basis for the vasoconstrictive activity of acellular Hb. Enhancing the molecular size of Hb by oligomerization (inter molecular cross-linking) and protein engineering the heme pockets of Hb by site directed mutagenesis to reduce the affinity of heme to NO have been the two major approaches that have been advanced as ways to overcome the vasoactivity of Hb.
Polyethylene glycol (PEG) chains have been used to modify Hb. The observation that PEGylated bovine Hb carrying ten copies of PEG-5000 linked to the surface amino groups of Hb through isopeptide linkage is vasoinactive, even though it has nearly the same affinity to NO as the parent Hb has suggested PEGylation of Hb is another approach to overcome or modulate the vasoactivity of acellular Hb without engineering the affinity of Hb to nitric oxide (16–18). The calculated molecular weight of this PEGylated bovine Hb is 104,000 kDa, and colligative properties (viscosity and colloidal oncotic pressure) of solutions of this PEGylated bovine are considerably higher than that of unmodified Hb. Accordingly, endowing Hb solutions with high viscosity and high oncotic pressure appeared to be a potential approach to overcome the hypertensive activity of acellular Hb. The molecular radius of the PEGylated bovine Hb is also higher than unmodifed Hb suggesting that the size enhancement of the Hb molecule that accompanies PEGylation may be another factor that has resulted in the neutralization of the vasoactivity of Hb. A polymeric form of ββ-sebacyl Hb with a molecular radius higher than 25 nm has also been shown to be non-hypertensive. Thus, the size enhancement that is accompanied by inducing high viscosity and colloidal oncotic pressure to Hb appears to be a potential new approach to overcome the vasoactivity without interfering with the NO binding activity of Hb.